Variable domain library and uses

Abstract

The invention provides polypeptides comprising a variant heavy chain variable framework domain (VFR). In some embodiments, the amino acids defining the VFR form a loop of an antigen binding pocket. In an embodiment, the polypeptide is a variable domain of a monobody and has a variant VFR. The polypeptide may optionally comprise one or more complementary determining regions (CDRs) of antibody variable domains. In an embodiment, the polypeptide is a variable domain of a monobody and has a variant VFR and one or more variant CDRs. Libraries of polypeptides that include a plurality of different antibody variable domains generated by creating diversity in a VFR, and optionally, one or more CDRs are provided and may be used as a source for identifying novel antigen binding polypeptides that can be used therapeutically or as reagents. The invention also provides fusion polypeptides, compositions, and methods for generating and using the polypeptides and libraries.

1. A polypeptide library comprising a plurality of monobody variable heavy chain domains, wherein each monobody variable heavy chain domain comprises a variant variable framework region (VFR), wherein the amino acid positions of the VFR form a loop in an antigen binding pocket, wherein the amino acid positions are according to the Kabat numbering system, wherein the VFR comprises:
 a) a N-terminal portion that comprises at least one structural amino acid position comprising one or both of the first two amino acid positions of the N-terminal portion of the VFR, wherein said structural amino acid position has a variant amino acid selected from the group consisting of up to six different amino acids, wherein the first amino acid position of the N-terminal portion corresponds to position 71 of a variable heavy chain domain and is C, F, Y, W, M, or L; 
 b) a central portion that comprises at least one nonstructural amino acid position, wherein the nonstructural amino acid position has a variant amino acid comprising any of the naturally occurring amino acids; and 
 c) a C-terminal portion that comprises at least one structural amino acid position, wherein said structural amino acid position has a variant amino acid selected from the group consisting of up to six different amino acids. 
 
     
     
       2. The polypeptide library of  claim 1 , wherein said at least one structural amino acid position of the C-terminal portion of the VFR is the last amino acid position of the C-terminal portion. 
     
     
       3. The polypeptide library of  1 , wherein said at least one structural amino acid position of the N-terminal portion corresponds to position 71 of a variable heavy chain domain and the variant amino acid is C, F, or Y, and wherein the amino acid position is according to the Kabat numbering system. 
     
     
       4. The polypeptide library of  claim 1 , wherein said at least one structural amino acid position of the N-terminal portion comprises both of the first two amino acid positions of the N-terminal portion, and wherein the first amino acid position corresponds to position 71 of a variable heavy chain domain and is C, F, or Y, and the second amino acid position corresponds to position 72 of a variable heavy chain domain and is D or E, and wherein the amino acid positions are according to the Kabat numbering system. 
     
     
       5. The polypeptide library of  claim 2  or  claim 4 , wherein said at least one structural amino acid position of the C-terminal portion corresponds to position 78 of a variable heavy chain domain and is C or F, and wherein the amino acid position is according to the Kabat numbering system. 
     
     
       6. The polypeptide library of  claim 1 , wherein the central portion is no more than 20 amino acids. 
     
     
       7. The polypeptide library of  claim 6 , wherein said at least one nonstructural position has a variant amino acid encoded by a non-random codon set. 
     
     
       8. The polypeptide library of  claim 1 , wherein said at least one structural amino acid position of the C-terminal portion corresponds to position 78 of a variable heavy chain domain and the variant amino acid is M, C, F, V, or I. 
     
     
       9. The polypeptide library of  claim 8 , wherein position 71 is a cysteine and position 78 is a cysteine. 
     
     
       10. The polypeptide library of  claim 1  wherein each monobody variable heavy chain domain is a fusion polypeptide. 
     
     
       11. The polypeptide library of  claim 10  wherein each fusion polypeptide is fused to at least a portion of a viral coat protein. 
     
     
       12. The polypeptide library of  claim 11 , wherein the viral coat protein is selected from the group consisting of p111, pv111, Soc, Hoc, 9pD, pV1 and variants thereof. 
     
     
       13. The polypeptide library of  claim 1 , wherein each monobody variable heavy chain domain further comprises a variant complementarity determining region 1 (CDR1) comprising:
 a) a N-terminal portion that comprises at least one structural amino acid position, wherein said structural amino acid position has a variant amino acid selected from the group consisting of up to six different amino acids; 
 b) a central portion that comprises at least one nonstructural amino acid position, wherein the nonstructural amino acid position has a variant amino acid comprising any of the naturally occurring amino acids; and 
 c) a C-terminal portion that comprises at least one structural amino acid position, wherein said structural amino acid position has a variant amino acid selected from the group consisting up to six different amino acids, and wherein the amino acid positions in the CDR1 region form a loop of the antigen binding pocket. 
 
     
     
       14. The polypeptide library of  claim 13 , wherein the structural amino acid position of the N-terminal portion of CDR1 corresponds to amino acid position 24 of a monobody variable heavy chain domain; the structural amino acid position of the C-terminal portion of CDR1 corresponds to amino acid position 34 of a monobody variable heavy chain domain; and the central portion amino acid positions of CDR1 correspond to amino acids positions 25 to 33 of a monobody variable heavy chain domain; and wherein the amino acid positions are according to the Kabat numbering system. 
     
     
       15. The polypeptide library of  claim 14 , wherein the structural amino acid position at position 24 is F, Y, V, or I and at position 34 is F, V, or I. 
     
     
       16. The polypeptide library of  claim 1 , wherein each monobody variable heavy chain domain further comprises a variant complementarity determining region 2 (CDR2) comprising:
 a) a N-terminal portion that comprises at least one structural amino acid position, wherein said structural amino acid position has a variant amino acid selected from the group consisting of up to six different amino acids; and 
 b) a central portion that comprises at least one nonstructural amino acid position, wherein the nonstructural amino acid position has a variant amino acid comprises any of the naturally occurring amino acids; and wherein the amino acid positions of the CDR2 form a loop of the antigen binding pocket. 
 
     
     
       17. The polypeptide library of  claim 16 , wherein the structural amino acid position of the N-terminal portion of CDR2 corresponds to amino acid position 51 of a variable heavy chain domain and the central portion amino acid positions of CDR2 correspond to amino acid positions 52 to 56 of a variable heavy chain domain and can be any of the naturally occurring amino acids; and wherein the amino acid positions are according to the Kabat numbering system. 
     
     
       18. The polypeptide library of  claim 17 , wherein the structural amino acid position at position 51 is F or L. 
     
     
       19. The polypeptide library of  claim 1 , wherein each monobody variable heavy chain domain further comprises a variant complementarity determining region 3 (CDR3) comprising:
 a) a N-terminal portion that comprises at least one structural amino acid position, wherein said structural amino acid position has a variant amino acid that is selected from the group consisting of up to six different amino acids; 
 b) a central portion that comprises at least one nonstructural amino acid position, wherein the nonstructural amino acid position has a variant amino acid that comprises any of the naturally occurring amino acids; and 
 c) a C-terminal portion that comprises at least one structural amino acid position, wherein said structural amino acid position has a variant amino acid that is selected from the group consisting of up to six different amino acids, and wherein the amino acid positions in the CDR3 region form a loop of the antigen binding pocket. 
 
     
     
       20. The polypeptide library of  claim 19 , wherein the structural amino acid positions of the N-terminal portion of CDR3 correspond to amino acid positions 96 and 97 of a variable heavy chain domain; the structural amino acid positions of the C-terminal portion of CDR3 correspond to amino acid positions 100i, 100j, 100k and 100l of a variable heavy chain domain; and the central portion amino acid positions of CDR3 correspond to amino acid positions 98 to 100h of a variable heavy chain domain; and wherein the amino acid positions are according to the Kabat numbering system. 
     
     
       21. The polypeptide library of  claim 20 , wherein amino acid position 96 is R, amino acid position 97 is I, amino acid position 100i is W, amino acid position 100j is V, amino acid position 100k is T, and amino acid position 100l is W. 
     
     
       22. A method of generating a plurality of monobody variable heavy chain domains, each monobody variable heavy chain domain comprising a variable framework region (VFR), wherein the amino acid positions of the VFR form a loop in an antigen binding pocket and, wherein the amino acid positions are according to the Kabat numbering system, said method comprising:
 a) replacing an amino acid in at least one structural amino acid position comprising one or both of the first two amino acid positions of the N-terminal portion of the VFR, with up to six different amino acids, wherein the first amino acid position of the N-terminal portion corresponds to position 71 of a variable heavy chain domain and is C, F, Y, W, M, or L; 
 b) replacing an amino acid in at least one nonstructural amino acid position with any naturally occurring amino acid, wherein the nonstructural amino acids comprise about 1 to 20 contiguous amino acids; and 
 c) replacing an amino acid in at least one structural amino acid position at the C terminus of the VFR with up to six different amino acids. 
 
     
     
       23. The method according to  claim 22 , wherein the monobody variable domain is a variable heavy chain domain is a variable domain of a camelid monobody.